ABSTRACT The cost and consequences of CNS disorders such as addiction, autism, anxiety, and depression place an enormous burden on American society. Despite significant progress, treatments for these disorders are limited and have many side effects. Thus safer, more specific drugs would have high clinical utility. One high level drug-able target is the metabotropic glutamate receptor, mGlu5, which plays a critical role in all of these disorders. Indeed, several mGlu5 negative allosteric modulators (NAMs) are currently in clinical trials with varying degrees of efficacy. Besides the chemical scaffold, drug efficacy is also determined by cellular properties and receptor location. Because our previous work has shown that 60-90% of mGlu5 is located on intracellular membranes where it couples to distinct signaling systems, mGlu5 location may play a key role in its ability to be modulated. In fact our new data show that the intracellular receptor is necessary for establishing hippocampal and striatal LTD and sufficient in blocking chronic pain behaviors. Caveats exist, though, since many of these initial antagonists have off- target effects, variable efficacy, and rapid clearance. To overcome these issues we have obtained five new highly selective mGlu5 NAMs from Eli Lilly, Inc. Our initial data included in this application indicate that at least one of these NAMs only blocks cell surface mGlu5 whereas others are freely permeable. Using pharmacological, molecular and genetic tools as well our unique combination of permeable and impermeable agonists and antagonists, here we propose to test whether these novel NAMs block cell surface or intracellular mGlu5 in the striatum and the hippocampus, regions relevant to many CNS disorders. NAMs that differentially regulate mGlu5 will be further tested in ex vivo models of synaptic plasticity. As postsynaptic mGlu5 activation is required for hippocampal and striatal endocannabinoid-mediated LTD, the proposed studies will also determine which receptor pool contributes to presynaptic endocannabinoid-LTD. New regulators like the NAMs tested here will 1) further the concept that the cellular context of receptor modulation may alter its efficacy; 2) determine whether intracellular mGlu5 underlies fundamental aspects of synaptic activity; and 3) provide a critical tool (s) for future in vivo CNS applications. At the therapeutic level, the occurrence of a functional intracellular receptor opens the door to selectively tailoring agonists and/or antagonists to either receptor pool. Future studies targeting drugs to cell surface versus intracellular receptors might lead to new therapeutic tools for addiction, autism, anxiety, and other mGlu5-modulated disorders.